Processes for coupling amino acids using bis-(trichloromethyl) carbonate

ABSTRACT

A process is disclosed for using bis-(trichloromethyl)carbonate (triphosgene), diphosgene or phosgene as efficient and effective coupling reagents during coupling of carbohydrates to peptide chains. This process is particularly useful for the coupling to sterically hindered amino acid residues, or for other difficult couplings. The reagents can be used for the derivatization of peptides by formation of a bond between a free amine on a peptide and a carbohydrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/756,223filed Jan. 9, 2001 now U.S. Pat. No. 6,512,092, which is a continuationof the U.S. national stage designation of International Application no.PCT/IL99/00378 filed Jul. 11, 1999, the content of which is expresslyincorporated herein by reference thereto.

FIELD OF THE INVENTION

The invention relates to a process for the in situ generation of aminoacid chlorides utilizing bis-(trichloromethyl)carbonate, commonly knownas triphosgene, and to methods of using this process for solid phasepeptide synthesis and for derivatization of a solid support.

BACKGROUND OF THE INVENTION

In the field of peptide synthesis certain couplings are known asdifficult couplings, especially those involving coupling to bulky orsterically hindered amino acid residues, such as N alkylated,C-alkylated and C^(α) branched amino acids. In order to obtainacceptable yields when these couplings are performed a variety ofspecial coupling reagents have been developed. Among other knownprocedures, is the use of pre-formed amino acid chlorides to improve theoutcome of the coupling reactions.

The general use of protected amino acid chlorides in solid phase peptidesynthesis (SPPS) is limited mainly because of the fact that chlorides offluorenylmethoxycarbonyl (Fmoc) amino acid having side chains protectedwith acid labile protecting groups, including but not limited to t-butyl(t-Bu), t-butoxycarbonyl (Boc) or trityl (Trt), have limited shelfstability. For example, chlorides of Fmoc-amino acids (AAs) witht-Bu-protected side chains could not generally be accommodated. In somecases (aspartic acid and glutamic acid) the chlorides could not beobtained and in other cases (tyrosine, serine, threonine) their shelfstability appeared insufficient for practical utilization. In addition,the preparation of chlorides derived from Fmoc-Lysine(Boc),Fmoc-Tryptophan (Boc), Fmoc-Cysteine(Trt), Fmoc-Glutamine(Trt) andFmoc-Arginine 2,2,5,7,8-Pentamethyl chroman-6-sulphonyl (Pmc) isproblematic because of side reactions and require special reactionconditions and purification (Carpino et al. Acc. Chem. Res. 29:268,1996). This problem also hampers the general use of pre-formed Fmocamino acid chlorides in automatic peptide synthesis. Despite theselimitations, acid chlorides were used in SPPS especially for theassembly of hindered secondary amino acids (see Carpino et al. 1996 ibidand refs. within).

Coupling of protected amino acids to N^(α)-alkylated amino acids waspreviously considered to be a difficult coupling both in solution andsolid phase. This coupling was used in model peptides to demonstrate theefficiency of new, more effective, coupling methods. In these models,N-Methylated amino acids were used as nucleophiles, since coupling toN-Methylated amino acids having steric hindrance on the C^(α) (e.g.,N-methyl valine and N-methyl Aminoisobutyric acid) was found to be muchslower than to proline. Certain coupling agents and activation methodssuch as bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP)(Coste et al. Tetrahetron Lett. 31 669, 1990),1-hydroxy-7-azabenzotriazole(HOAt)/O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (Carpino et al. J. Chem. Soc., Chem. Commun.201, 1994), urethane-protected N-carboxyanhydrides (UNCA) (Spencer etal. Int. J. Pep. Prot. Res. 40:282, 1992) and acid halides (Carpino etal., 1996 ibid) were specially recommended to achieve coupling toN-alkyl amino acids.

The acid chloride method was found to be a superior way to coupleprotected amino acids to sterically hindered amino acid derivatives,such as the N-alkyl amino acids during SPPS of backbone cyclic peptides.To overcome the limitations of the pre-formed acid chloride method andto allow its general use in SPPS, it would be advantageous to have anefficient and generally applicable method allowing the in-situgeneration of Fmoc-AAs chlorides.

The reagent bis-(trichloromethyl)carbonate (BTC) (Councler, C. Ber.Dtsch. Chem. Ges. 13:1697, 1880) also named hexachlorodimethyl carbonateor “triphosgene” is a solid stable phosgene substitute equivalent tothree moles of phosgene. Triphosgene has been used as an efficientcarbonylating agent for liquid and solid phase synthesis of variousaza-analogues of peptides containing aza-alanine, aza-aspartic acid andaza-asparagine residues (Andre et al. J. Pep. Sci. 3:429, 1997).

The use of triphosgene as a reagent for formation of isocyanates orother reactive species useful in peptide chemistry has also beendisclosed (Eckert DE3440141, Nippon Kayaku JP10007623). The usefulnessof triphosgene in preparation of various intermediates forpharmaceuticals has also been disclosed (Hoffmann et al. DD292452).

It is neither taught nor suggested in the art that the triphosgenereagent is suitable for the in-situ generation of protected amino acidchlorides, namely as a coupling agent in SPPS (for review see Cotarca etal. Synthesis 553, 1996).

Phosgene gas has long been a valuable asset to both lab and plant scaleoperations however the dangers of using it are also well documented,especially the respiratory hazards. Liquid trichloromethylchloroformate, commonly known as “diphosgene” (Fridgen, L. N. and Prol,J. J., J. Org. Chem. 54:3231, 1989) which has already been used as aphosgene substitute, has proven useful in all common phosgene reactions,but being a liquid its transport and storage still impose considerablehazard. Being a crystalline solid (mp 81–83° C.), BTC is safer and easyto handle and therefore became the reagent of choice for allapplications where phosgene chemistry is required (Cotarca et al. ibid).Synthetically, one mole of BTC yields three mole-equivalents of phosgenewhich reacts with hydroxyl, amine or carboxylic acid nucleophilesforming chloroformate, isocyanate or acyl chloride, respectively.

Considering all these features together with the fact that BTC isinexpensive and less susceptible to hydrolysis than phosgene, it issurprising that the use of BTC as a general coupling agent has not beenconsidered.

Backbone Cyclized Peptide Analogs

Backbone cyclization is a concept that allows the conversion of peptidesinto conformationally constrained peptidomimetics with desiredpharmacological properties such as metabolic stability, selectivity andimproved bioavailability (Gilon et al. Biopolymers 31:745, 1991; Byk etal. J. Med. Chem. 39:3174, 1996; Gilon et al. J. Med. Chem. 41:919,1998).

In backbone cyclization the N^(α) and/or C^(α) atoms in the peptidebackbone are linked through various spacers. To synthesize N-backbonecyclic peptides a large number of orthogonally protected-functionalizedN^(α) alkyl amino acids (N-building units) were prepared (Bitan et al.J. Chem. Soc., Perkin trans. I 1501, 1997a; Muller et al. J. Org. Chem.62:411, 1997). These units were incorporated into peptides by SPPS orsolution methodologies and after orthogonal removal of the protectinggroups from the ω-functions on the N^(α)-alkyl they are cyclized. Acritical step in the synthesis of N-backbone cyclic peptides is thecoupling of protected amino acids to the sterically hindered secondaryamine of the N^(α) (ω-functionalized alkyl) amino acid residue on thepeptidyl-resin.

The synthesis of N-backbone cyclic peptides that incorporate N^(α)(ω-functionalized alkyl) Glycine building units were reportedpreviously. In these cases couplings of the protected amino acids to thesecondary amine of the Gly building unit attached to peptidyl resin wereachieved by multiple couplings withbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexaflourophosphate BOP or PyBroP as coupling agents (Bitan et al. J.Pept. Res. 49:421, 1997b; Byk et al., 1996 ibid).

Generally, the coupling of protected AAs to building units other thanGly (non-Gly backbone cyclic building units) were found to be difficultand even impossible.

It has been shown that the coupling of many Fmoc AAs to stericallyhindered secondary amines including a variety of non-Gly building unitsattached to peptidyl-resin could be achieved in moderate to high yieldsusing the acid-chloride method but not acid fluorides (Carpino et al.1996 ibid) or other coupling agents such as PyBrOP (Coste et al., ibid),HOAt/HATU Carpino 1994 ibid),2-(2-Oxo-1(2H)-pyridyl)-1,1,3,3-bispenta-methyleneuroniumtetrafluoroborate (TOPPipU) (Henkleinet al. In ““Peptides 1990” Proc. ofthe 21th European Peptide Symposium”, E. Giralt and D. Andreu, eds, pp.67. ESCOM Leiden, 1990), UNCA (Spencer et al., 1992) and Mukaiyamareagent (Mukaiyama, T. Angew. Chem., Int. Ed. Ingl. 18:7078, 1979).

SUMMARY OF THE INVENTION

The present invention relates to a process for the improvement ofdifficult couplings in SPPS. The invention also provides a method forimproving the yield of the desired stereoisomer in solid phase peptidesynthesis. In addition, the invention further provides methods forfacilitating multiple parallel synthesis (MPS).

The present methods are useful for attaching protected amino acids tofunctionalized solid supports or to attach biomedically importantligands to a peptide or peptidyl resin during SPPS. Furthermore, thesemethods can be used to cyclize peptides attached to a solid support, orto form a urea bond in the sequence or in the bridge of cyclizedpeptides.

According to the present invention, a process is provided for the insitu generation of protected amino acid chlorides, by use of an agentsuch as phosgene, diphosgene or more preferably triphosgene. Theprotected amino acid chlorides thus generated are particularly useful inthe coupling of an amino acid residue to a peptide chain. They can alsobe used for the coupling of a carbohydrate moiety to a peptide chain.

The in situ generation of acid chlorides using the methods of thepresent invention thus further provides a process whereby otherbiologically important acids, including but not limited to glucoronicacid, DTPA, and DOTA, may be connected to a peptide chain through theamine backbone or through an amino acid side chain functionality.

It has now been found that in accordance with the principles of thepresent invention bis-(trichloromethyl)carbonate, also known by thetrivial chemical name triphosgene, can be used for the in situgeneration of protected amino acid chlorides. This process overcomesmany of the problems encountered in difficult coupling steps in SPPS,particularly where the coupling step involves sterically hindered aminoacid residues or bulky amino acid analogs.

Methods are provided for the use of BTC as a convenient and efficientcoupling agent for difficult couplings in SPPS. These methods providegreatly enhanced yields of the desired product in SPPS with retention ofconfiguration and without undesired side reactions and also facilitatethe performance of multiple parallel peptide synthesis. These methodsalso facilitate the synthesis of complex peptide analogs with multiplecyclizations, e.g., bi- and tri-cyclic peptides.

One method according to the present invention provides a process ofcoupling an amino acid residue to a peptide chain comprising:

(i) providing an amino acid residue having a free carboxylic group andblocked amino group, optionally having additional blocked functionalside chains;

(ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonatein an solvent inert to this reaction to obtain an amino acid chloride;

(iii) neutralizing the free acid by addition of an organic base;

(iv) adding the resulting suspension containing the amino acid chlorideto a compound selected from the group consisting of a peptide having ablocked carboxyl terminus and a free amino terminus, and a peptidylresin having at least one free amino terminus;

(v) providing reaction conditions enabling the coupling of the aminoacid chloride to the peptide to yield a peptide elongated by one aminoacid residue.

A second method according to the present invention provides a process ofcoupling an amino acid residue to a solid support comprising:

(i) providing an amino acid residue having a free carboxylic group andblocked amino group, optionally having additional blocked functionalside chains;

(ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonatein a solvent inert to the reaction to obtain an amino acid chloride;

(iii) neutralizing the free acid by addition of an organic base;

(iv) adding the resulting suspension containing the amino acid chlorideto a compound selected from the group consisting of a resin having atleast one free amino terminus and a solid support having a functionalgroup capable of binding the chloride;

(v) providing reaction conditions enabling the coupling of the aminoacid chloride to the solid support.

A currently most preferred embodiment according to the present inventionis summarized below in Scheme 1:

-   -   BTC=Bis-(trichloromethyl)carbonate (triphosgene)    -   Fmoc-AA=Fluorenylmethoxycarbonyl-proteinogenic-α-Amino Acid    -   R=Side chains of all proteinogenic α-amino acids    -   R′=CH3, (CH2)_(n=2-4)-NH-Alloc, (CH2)_(n=2-3)-COOAllyl    -   R₃″N=tertiary or aromatic amine

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. HPLC chromatogram of the product obtained in example 38.

FIG. 2. Mass spectra analysis of the product obtained in example 38.

FIG. 3. HPLC chromatogram of the product obtained in example 49.

FIG. 4. HPLC chromatogram of the product obtained in example 55.

FIG. 5. HPLC chromatogram of PTR 3227 obtained in example 63.

FIG. 6. Mass spectra analysis of PTR 3229 obtained in example 64.

FIG. 7. Mass spectra analysis of PTR 3237 obtained in example 65 A.

FIG. 8. HPLC chromatogram and mass spectra analysis of PTR 3241 obtainedin example 65 B.

FIG. 9. HPLC chromatogram and mass spectra analysis of MPS peptidenumber 20, obtained in example 66.

FIG. 10. HPLC chromatogram and mass spectra analysis of a Cyclosporineanalog synthesized in example 67.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention processes for the in situ generationof protected amino acid chlorides are provided utilizing an agent suchas phosgene, diphosgene or triphosgene. In all the specific preferredembodiments presented herein the processes are exemplified utilizingsolid phase peptide synthesis although these processes are also highlysuitable to the synthesis of peptides in solution as known in the art.Moreover, the same processes can potentially be used to the coupling ofother moieties to a peptide chain, including but not limited to thecoupling of a carbohydrate moiety to a peptide chain, and for themethods of multiple parallel peptide synthesis. The processes of thepresent invention are also highly suitable to the synthesis of complexpeptide analogs such as bi- and tri-cyclic peptides, and synthesis ofCyclosporine analogs which comprise seven sterically hindered N-methylamino acids. The processes are also suitable for formation of a ureabond in the sequence or in the bridge of cyclized peptides.

Abbreviations

Certain abbreviations are used herein to describe this invention and themanner of making and using it.

For instance, AA refers to amino acid; ACN refers to acetonitrile; Allocrefers to Allyloxycarbonyl; Boc refers to t-butoxycarbonyl; BOP refersto benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphoniumhexaflourophosphate; BTC refers to bis-(trichloromethyl)carbonate; DCMrefers to dichloromethane; DIEA, diisopropylethylamine; DOTA refers totetraazacyclodecanetetraacetic acid; DTPA, diethylenetriaminepentaaceticacid; eq refers to equivalents; Fmoc refers to fluorenylmethoxycarbonyl;Gln refers to Glutamine; HATU,O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HPPA,parahydroxy phenyl propionic acid; MBHA refers to methylbenzhydrilamine;Me refers to methyl; MPS refers to multiple parallel synthesis; NMPrefers to N-methyl pyrollidone; Pmc refers to 2,2,5,7,8-Pentamethylchroman-6-sulphonyl; PyBrOP, bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate; SPPS refers to solid phase peptide synthesis; t-Burefers to t-butyl; TFA refers to trifluoroacetic acid; THF refers totetrahydrofuran; TIS refers to triisopropylsilane; TOPPipU refers to2-(2-Oxo-1(2H)-pyridyl)-1,1,3,3-bispenta-methyleneuroniumtetrafluoroborate; Trt refers to trityl; UNCA refers tourethane-protected N-carboxyanhydrides.

Amino Acids

The amino acids used in this invention are those which are availablecommercially or are available by routine synthetic methods. Naturalcoded amino acids and their derivatives are represented by three-lettercodes according to IUPAC conventions. When there is no indication, the Lisomer was used. The D isomers are indicated by “D” before the residueabbreviation.

List of Non-coded amino acids: Abu refers to 2-aminobutyric acid, Aibrefers to 2-amino-isobutyric acid, β-Ala refers to β-Alanine, Amb refersto 3 Amino methyl benzoic acid; ChxGly refers to cyclohexyl Glycine, Dabrefers to Di amino butyric acid, GABA refers to gamma amino butyricacid, Hcys refer to homocystein, 1Nal refers to 1-naphthylalanine, 2Nalrefers to 2-naphtylalanine, Nva refers to norvaline, (p-Cl)Phe refers topara chloro Phenylalanine, (p-NH₂)Phe refers to para aminoPhenylalanine, (p-F)Phe refers to para fluoro Phenylalanine, (p-NO₂)Pherefers to para nitro Phenylalanine, Sar refers to Sarcosine, Thi refersto thienylalanine.

As used herein and in the claims, “blocked amino acid” denotes an aminoacid in which a reactive group is protected by a specific blocking orprotecting group which can be removed selectively, and may alternativelybe denoted by the term “protected amino acid” as well known in the art.

As used herein “backbone cyclic peptide” denotes an analog of a linearpeptide which contains at least one building unit that has been linkedto form a bridge via the alpha nitrogen of the peptide backbone toanother building unit, or to another amino acid in the sequence. A“building unit” indicates an N^(α)-ω-functionalized derivative of aminoacid.

Building Units Used in the Synthesis of Backbone Cyclized Peptides

A “building unit” indicates an N^(α)-ω-functionalized derivative ofamino acid having the general Formula No. 1:

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain, optionally bound with aspecific protecting group; B is a protecting group selected from thegroup consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls;and G is a functional group selected from the group consisting ofamines, thiols, alcohols, carboxylic acids and esters, aldehydes,alcohols and alkyl halides; and A is a specific protecting group of G.

Within the peptide sequence the building unit incorporated will have thefollowing structure:

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain, optionally bound with aspecific protecting group; and G is a functional group selected from thegroup consisting of amines, thiols, alcohols, carboxylic acids andesters, and alkyl halides; which is incorporated into the peptidesequence and subsequently selectively cyclized via the functional groupG with one of the side chains of the amino acids in said peptidesequence or with another ω-functionalized amino acid derivative.

The building units are abbreviated by the three letter code of thecorresponding modified amino acid followed by the type of reactive group(N for amine, C for carboxyl), and an indication of the number ofspacing methylene groups. For example, GlyC2 describes a modified Glyresidue with a carboxyl reactive group and a two carbon methylenespacer, and PheN3 designates a modified phenylalanine group with anamino reactive group and a three carbon methylene spacer.

The methodology for producing the building units is described ininternational patent applications published as WO 95/33765 and WO98/04583 and in U.S. Pat. Nos. 5,723,575 5,811,392; 5,883,293; 5,874,529and 5,770,687, all of which are expressly incorporated herein byreference thereto as if set forth herein in their entirety.

General Procedures:

For the majority of examples which follow, certain procedures were usedthroughout, which have therefore, for the sake of simplicity, beensummarized herein under the heading general procedures.

General Procedure A: SPPS of Model Peptides

1 g Rink Amide methylbenzhydrilamine (MBHA) resin (0.55 mmol/g) wereswelled for 1.5 hour in N-methyl pyrollidone (NMP) in a reaction vesselequipped with sintered glass bottom and placed on a shaker. The Fmocprotection was removed with 20% piperidine in NMP (twice fifteenminutes, 8 mL each). After washing with NMP (5 times, 8 mL for 2minutes), the Fmoc removal was monitored by Kaiser test.

A coupling cycle was carried out with Fmoc AA or Fmoc N-Me AA orFmoc-building unit (3 equivalents) PyBrOP (3 equivalents)diisopropylethylamine (DIEA) (6 equivalents) in NMP (8 mL) for 1 hour atroom temperature. Reaction completion was monitored by Kaiser test andthe solvent was removed by filtration. The resin was washed with NMP (5times, 8 mL 2 minutes). The Fmoc protecting group was removed as above.Coupling of Fmoc AA or Fmoc N-Me AA or Fmoc-building unit to AA-resinwas performed as described above, using PyBrOP as coupling agent.Coupling of Fmoc AA or Fmoc N-Me AA or Fmoc-building unit to N-Me orbuilding unit-resin was performed as described in General procedure B.Peptide elongation was performed by repeating the removal of the Fmocprotecting group and the coupling cycle described above. Final Fmocdeprotection was followed by washes (NMP 5 times, 8 mL 2 minutes anddichloromethane (DCM) 3 times, 8 mL 2 minutes). The peptidyl resin wasdried in vacuum. Fast cleavage: a small amount of peptidyl resin wastreated with 95% trifluouroacetic acid (TFA) containing 1%triisopropylsilane (TIS) and 4% H₂O. The solvents were removed by astream of nitrogen and the residue was taken up in water:acetonitrile(ACN containing 0.1% TFA). After filtration the solution was injected toHPLC and/or to the mass-spectrometer.

General Procedure B: Coupling of Fmoc-AA's to AA Peptidyl Resin and toN-alkylated AA Peptidyl Resin Using BTC

Fmoc AA or Fmoc N-Me AA or Fmoc-building unit (5 eq, 0.275 mmol) and BTC(1.65 eq, 0.09 mmol) were dissolved in either tetrahydrofuran (THF),dioxane, diglyme, or in 1,3-dichloropropane to give 0.15 M solution, towhich 2,4,6-collidine (14 eq, 0.75 mmol) was aded to give a whitesuspension. This suspension was poured into the N-Me AA or buildingunit's—peptidyl-resin prewashed with the appropriate solvent. Themixture was shaken at 50° C. for 1 hour and filtered. The peptidyl resinwas washed with DCM, swelled with the appropriate reaction solvent, andthe coupling repeated once more. In cases where coupling was performedwith Fmoc-AA's to AA peptidyl resin, only 3 eq of Fmoc-AA's, 1 eq of BTCand 8 eq of 2,4,6-collidine were used in dioxane at room temp for 1 h.

General Procedure C: Coupling of Fmoc-AA or N-Me AA or Building Unit toa Functionalized Solid Support

Fmoc AA or Fmoc NMeAA or Fmoc-building unit chloride was prepared insitu as described in Procedure B. The suspension in dioxane was pouredonto preactivated glass or onto hydroxymethylated polystyrene. Themixture was shaken at 50° C. for 1 hour and filtered. The derivatizedsupport was washed with DCM. The degree of derivatization was determinedby the Fmoc piperidine method.

General Procedure D: Derivatization of Amino or Hydroxy Peptidyl Resin

Acids such as 1,2,3,4-tetra-O-acetyl glucoronic acid,diethylentriaminepenta acetic acid (DTPA),tetraazacyclodecanetetraacetic acid (DOTA) or parahydroxy phenylpropionic acid (HPPA) were converted to their corresponding acidchlorides using BTC as described in procedure B. The suspension waspoured into amino or hydroxy peptidyl resin and heated 1 h. at 50° C.After filtration and wash, the derivatized peptidyl resin was cleavedand characterized as described in procedures A, G and H.

General Procedure E: SPPS of Backbone Cyclic Peptides—Cyclization andCleavage

Peptidyl-resins that contain two building units were synthesizedaccording to General procedures A & B. The Allyl/Alloc protecting groupswere removed by reaction with Tetrakis/triphenylphosphine)-palladium,acetic acid 5% and N-methylmorpholine 2.5% in DCM under argon, for 1.5hours at room temperature. The peptide resin was washed with chloroformfollowed by NMP. Cyclization was carried out in NMP with PyBOP (3equivalents) and DIEA (6 equivalents) for 1 hour at room temperature.After washings with NMP the cyclization was repeated. The backbonecyclic peptide was deprotected and cleaved from the resin by treatmentwith 10 mL of TFA 94%, water 2.5%, TIS 1% and ethanedithiol 2.5% at 0°C. under argon for 0.5 hour and at room temperature for 1–3 hours. Theresin was removed by filtration and washed with additional amount ofTFA, the combined solution evaporated by a nitrogen stream to give anoil which upon treatment with cold ether (40 mL) solidified. The etherremoved after centrifugation and the solid dried in high vacuumovernight to give the crude peptide.

General Procedure F: Cyclization of Peptides Containing Two N-buildingUnits Using BTC

Peptidyl-resins that contain two N-building units were synthesizedaccording to General procedures A, B and E. Cyclization was carried outin dioxane with BTC (0.33 eq) at room temp' for 1 h. Chemical proceduresand characterization were done as described in General Procedure E, Gand H.

General Procedure G: HPLC Analysis of Crude Peptides

A sample of the crude peptides was dissolved in solvent A (water+0.1%TFA) and injected into the HPLC machine (column C18 250×4 mm, flow 1mL/minute). Eluent solvent A and B (ACN, 0.1% TFA). Hydrophobic peptideswere eluted using linear gradient from 90% to 10% A in 35 minutes.Hydrophilic peptides were eluted using linear gradient from 100% to 10%A in 35 minutes. Peptides were detected by an online UV detector set at214 nm.

General Procedure H: Mass Spectral Analysis of Peptides

Crude peptides or fractions collected from the HPLC were analyzed byquadrupole or ion trap mass spectrometers.

The invention will be exemplified with regard to particular peptides andpeptidomimetic compounds. These examples are to be construed in anon-limitative fashion, and it is understood that the invention is notlimited by the scope of the examples, but rather by the scope of theclaims which follow the specification.

EXAMPLES

Carboxylic acid chlorides are most conveniently prepared when BTC isreacted with carboxylic acids in an solvent inert to BTC at temperaturesranging from slightly above room temperature to reflux temperature,preferably in the presence of DMF or tertiary amines as catalysts. It isof importance that the solvent used be inert to BTC in order to obtainthe desired synthetic results.

With that in mind and the fact that in the case of the pre-preparedFmoc-AA's chlorides best conversions were obtained by heating in NMP,our preliminary studies with BTC were conducted under the followingconditions: Fmoc-AA's (3 eq) and BTC (1.2 eq) were reacted in NMPfollowed by DIEA (12 eq) addition. After half an hour at roomtemperature, the activated AA's were poured into the peptide resin whichwas swelled at 60° C. for 1 h. Double coupling under these conditionsgave the desired peptides but unfortunately with the loss ofstereochemistry. Changing the base to the weaker sterically hindered2,4,6-collidine did not improve the coupling stereointegritysignifically, as shown in Table 1.

It is concluded that the solvent NMP while being very efficient ingeneral procedures of solid phase peptide synthesis as are well known inthe art is not a suitable reagent for BTC mediated couplings.

TABLE 1 Summary of difficult couplings using BTC/NMP at 65° C. R.T. %Conversion Product Mass Ratio of No. Substrate (min) Product (a) Calc.Found R.T. (min) (b) isomers (c) 1 AlaN3-Thr-Rink 24.3Arg-Val-AlaN3-Thr-NH₂ 100 585.41 586.83 20.42 + 20.81 1:9 2AlaN3-Thr-Rink 24.3 Arg-Phe-AlaN3-Thr-NH₂ 100 633.41 634.86 24.10 +25.92 1:1 3 AlaC3-Thr-Rink 6.20 Phe-AlaC3-Thr-NH₂ 100 462.29 463.114.23 + 14.65 1:3 4 AlaC3-Thr-Rink 6.20 Leu-AlaC3-Thr-NH₂ 100 428.31429.1 12.99 + 13.33 1:5 5 ValC3-Thr-Rink 7.21 Phe-ValC3-Thr-NH₂ 100490.32 491.1 17.04 + 17.42 9:1 6 ValC3-Thr-Rink 7.21 Leu-ValC3-Thr-NH₂100 456.34 457.1 16.04 + 16.31  1:20 7 Leu3-Thr-Rink 9.45Phe-LeuC3-Thr-NH₂ 95 504.34 505.1 19.70 + 20.09 1:3 8 Leu3-Thr-Rink 9.45Leu-LeuC3-Thr-NH₂ 90 470.36 471.1 18.79 one isomer 9 ValN3-Trp-Rink14.91 Phe-ValN3-Trp-NH₂ 20 590.06 591.3 19.87 one isomer 10ValN3-Trp-Rink 14.91 Leu-ValN3-Trp-NH₂ 20 556.39 557.2 19.72 one isomerIn entry 1–2, DIEA was used as base. R.T of substrate is for theFmoc-protected derivative. Entries 3–10 were performed in MPS format in5.5 mM scale with collidine as base. (c) Based on integration of areaunder the curve obtained from HPLC analysis. (b) Analyzed according togeneral procedure G for hydrophobic peptides. (c) Which have identicalmass but different retention times.

To explain the racemization of Fmoc AA's during their efficient BTCmediated coupling to building unit-peptidyl-resins in NMP we suggest amechanism (Scheme 2) in which excess NMP reacts with BTC to form 1 whichby losing phosgene and CO₂ furnished Vilsmayer-type intermediate 2.Addition of the optically pure Fmoc-AA to the chloroiminium ion 2, andelimination of the chloride, yields active ester 3, which couples to thepeptide resin. On the other hand, N-alkoxycarbonyl protected aminoactive ester 3 under base catalysis is known to transform to oxazolone 4which is ready racemizes. Further experiments with sterically hindered,polar amino acids, bearing protected side chains gave insufficientresults as shown in Table 2.

TABLE 2 Summary of experiments using BTC/NMP with polar amino acids %Product Mass R.T. (min) No. Fmoc-AA Substrate Conversion Calc. Found (d)11 Arg (Pmc) AlaC3-Thr-Rink 30(a)–100(b) 471.23 472.3 8.07 12 Glu (tBu)LeuC3-Thr-Rink 10(a)–50(b) 486.32 487.0 14.60 13 Glu (tBu)AlaC3-Thr-Rink 30 (c) 444.27 445.8 9.50 14 Gln (Trt) AlaC3-Thr-Rink 30(c) 443.28 444.9 8.52 15 Met AlaC3-Thr-Rink 20 (c) 446.27 447.9 13.08 16Trp (Boc) AlaC3-Thr-Rink 80 (c) 501.3 502.8 16.22 (a) After 1 h. at 75°C.; (b) After 3 coupling cycles at 65° C.; (c) After 2 coupling cyclesat 65° C.; (d) Analyzed according to the general procedure G forhydrophobic peptides.

The total failure of these amino acids and a large variety of otherpolar and aromatic Fmoc AA's to couple to the more hindered buildingunits-peptidyl-resin, and also the fact that no reaction was obtainedwith Fmoc-Asn(Trt) and Fmoc-His(Trt) even with Ala-buildingunit-peptidyl-resin in NMP, required changing to a solvent inert to thereaction. Indeed, double coupling of Fmoc-Val and Fmoc-Ile toN^(α-)(ω-carballyloxypropyl)-Ala (denoted AlaC3) andN^(α-)(ω-carballyloxypropyl)-Leu (denoted LeuC3)-peptidyl-resins usingBTC in THF for only 1 h. at 50° C. afforded the desired peptide in 100%conversion and with no detectable racemization. These results promptedus to undertake a wide synthetic effort which includes the coupling ofall proteinogenic Fmoc-AA's (except Gly) to a large variety of buildingunits-peptidyl-resins and also to N-Me-Ala and toN-Me-Phe-peptidyl-resins, where the size and sequence of the peptidylmoiety varies. The results are shown in Table 3 and Table 4.

TABLE 3 Summary of difficult coupling using BTC/THF, Dioxane, Diglyme orDCP % Fmoc Con- Product Mass R.T. No AA Substrate vers Calc. Found (min)17 Ala AlaN3-Rink 100 502.9^(A) 503.9^(A) 9.45^(AX) A,B 583.4^(B)584.4^(B) 11.65^(BY) 18 Ala AlaC2-Rink 100 554.1 555.1 13.92^(Y) 19 AlaAlaC3-Rink 100 682.4^(A) 683.4^(A) 14.13^(AY) A,B 633.5^(B) 634.5^(B)13.37^(BY) 20 Ala AlaN2-Rink 100 569.3^(A) 570.3^(A) 11.01^(AY) A,B488.16^(B) 489.2^(B) 9.79^(BX) 21 Ala LeuN4-Ser-Rink 90 443.15 444.913.30^(X) 22 D-Ala AlaN2-Peptide- 100 682.4 683.4 14.13^(Y) Rink 23Arg(Pmc) PheC2-Thr-Rink 94 533.33 534.3 24.38^(Y) 24 Asp(tBu)LysC3-Thr-Rink 100 487.31 488.9 15.67^(Y) 25 Cys(Trt) ValC3-Thr-Rink 100446.26 447.1 26.39^(Y) 26 Glu(tBu) LysC3-Thr-Rink 70 501.33 502.216.81^(Y) 27 Gln(Trt) PheC2-Thr-Rink 100 505.28 506.2 28.97^(Y) 28Gln(Trt) ValC3-Thr-Rink 82 471.2 472.2 22.95^(Y) 29 Gln(Trt)LeuN4-Ser-Rink 100 500.9 501.9 12.34^(X) 30 D-Leu LeuN4-Ser-Rink 100486.0 487.0 17.70^(X) 31 Leu PheN2-Gln-Rink 100 1081.7 1082.7 17.31^(X)32 Leu LysC3-Thr-Rink 100 485.37 486.2 22.43^(Y) 33 Ile LeuC3-Thr-Rink80 470.36 472.0 18.97^(X) 34 Lys(Boc) LeuN4-Ser-Rink 100 501.0 502.011.26^(X) 35 Lys(Boc) PheC2-Thr-Rink 100 505.32 506.1 24.53 36 Lys(Boc)ValC3-Thr-Rink 56 472.2 473.0 22.12^(Y) 37 Lys(Boc) PheN2-Rink 1001002.4 1003.4 23.21^(X) 38 D-Lys(Boc) PheN2-Rink 100 1002.4 1003.423.02^(X) 39 D-Lys(Boc) PheN2-Rink 100 968.4 969.5 22.71^(X) 40 MetLeuN4-Ser-Rink 100 503.15 504.9 16.08^(X) 41 Phe AlaC3-Peptide- 1001716.0 1716.8 14.76^(X) Rink 42 Phe AlaN2-Rink 100 678.49 679.920.45^(X) 43 Phe N-MePhe-N- 100 528.2 529.9 22.06^(Y) MePhe-Rink 44PheC1 N-MeAla-Ala- 100 616.8 617.8 16.25^(X) AlaN2-Rink 45 PheC2N-MeAla-Ala- 100 502.9 503.9 9.45^(X) AlaN3-Rink 46 PheC2 N-MeAla-Ala-100 488.2 489.2 9.79^(X) AlaN2-Rink 47 N-MePhe N-MePhe-Rink 100 528.2529.9 22.06^(Y) 48 N-MePhe N-MePhe-N- 100 500.18 501.2 18.52^(X)MePhe-Rink 49 Pro ValC3-Thr-Rink 100 440.31 441.2 25.95^(Y) 50 Ser(tBu)LysC3-Thr-Rink 100 459.32 460.1 15.61^(Y) 51 Thr(tBu) AlaC2-Peptide- 1001587.4 1588.7 19.25^(Y) Rink 52 Thr(tBu) AlaC3-Peptide- 100 1601.41602.7 19.67^(Y) Rink 53 Thr(tBu) ValC3-Thr-Rink 53 444.3 445.124.35^(Y) 54 Trp(Boc) ValN3-Trp-Rink 100 629.38 630.2 20.01^(X) 55Trp(Boc) ValC3-Thr-Rink 100 529.33 530.1 17.80^(X) 56 Tyr(tBu)LeuN3-Amb-Ala- 100 1322.6 1323.6 19.05^(X) Arg-Rink 57 D-Tyr(tBu)AlaN3-Rink 100 1716.0 1716.8 14.76^(X) 58 Val AlaC3-Thr-Rink 100 414.29416.0 11.93^(X) 59 Val LysC3-Thr-Rink 92 471.35 472.1 19.49^(Y) 60 ValValC3-Thr-Rink 66 442.32 443.1 14.94^(X) 61 PheC3 Thr-Rink 100 391.23392.9 10.64^(X)Product Mass Data:

In entries 17, 18, 19, 20, 22, 31, 37, 44, 45, 46, 57 data correspond tothe cyclic peptides.

In entries 42, 51, 52, 56 data corresponds to the Allyl/Alloc protectedpeptides.

In entries 22, 41, 48, 51, 52 data correspond to the linear peptides.

In entries 43, 47 data correspond to the Ac-N tripeptide.

Peptide Sequences:

17^(A)) PheC2-NMeAla-Ala-AlaN3-NH₂;

17^(B)) GlyC1-Ala-Lys-(D)Ala-Ala-AlaN3-NH₂;

18) Ala-Ala-Lys-(D)Ala-Ala-AlaC2-NH₂;

19^(A)) (D)Ala-AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3-NH₂;

19^(B)) AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3-NH₂;

20^(A)) GlyC1-Ala-Lys-(D)Ala-Ala-AlaN2-NH₂;

20^(B)) PheC2-N-MeAla-Ala-AlaN2-NH₂;

22) (D)Ala-AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3-NH₂;

31) TrpC3-Ser-Glu-Tyr-Leu-PheN2-Gln-NH₂ (SEQ ID NO:1);

37) PheC1-Phe-Phe-(D)Trp-(L)Lys-PheN2-NH₂;

38) PheC1-Phe-Phe-(D)Trp-(D)Lys-PheN2-NH₂;

39) PheC1-Phe-Leu-(D)Trp-(D)Lys-PheN2-NH₂;

41) Biotin-Trp-Arg-Lys-(D)Arg-Phe-AlaC3-NH₂;

42) PheC1-Ala-Phe-AlaN2-NH₂;

47) Phe-NMePhe-NMePhe-NH₂;

48) NMePhe-NMePhe-NMePhe-NH₂;

51) Thr-AlaC2-Ser-Glu-Asn-His-Leu-Arg-His-Ala-LeuN3-Ser-NH₂ (SEQ IDNO:2);

52) Thr-AlaC3-Ser-Glu-Asn-His-Leu-Arg-His-Ala-LeuN3-Ser-NH₂ (SEQ IDNO:3);

56) TrpC3-Ser-Glu-Tyr-LeuN3-Amb-Ala-Arg-NH₂ (SEQ ID NO:4); and

57) Biotin-Trp-Arg-Lys-(D)Arg-Phe-AlaC3-Leu-Arg-(D)Tyr-AlaN3-NH₂.

HPLC Analysis Methods:

^(X) Analyzed according to the general procedure G for hydrophobicpeptides.

^(Y) Analyzed according to the general procedure G for hydrophilicpeptides.

As can be seen in Table 3 the conversion of most of the couplings werequantitative regardless of the incoming Fmoc-AAs, the structure of thebuilding unit, the sequence and the size of the peptidyl moiety. In thefollowing description the bold numbers in brackets denote the examplenumbers, as per table 3.

Three peptides (26, 36 and 53) gave below 70% conversion. It should benoticed that these couplings were not optimized.

Contrary to the pre-formed acid chloride method, where there was a majorproblem with the lability of the side chain protection groups, using themethods of the current invention the coupling of the following Fmoc-AAsgave substantially complete conversion: Arg(Pmc) (23), Asp(t-Bu) (24),Cys(Trt) (25), Lys(Boc) (34, 35), Ser(t-Bu) (50), Thr(t-Bu) (51, 52),Trp(Boc) (54) and Tyr(t-Bu) (56, 57). Since the side-chain Bocprotecting group is easily removed by acids, we have monitored itsstability by the Keiser test during the BTC mediated coupling ofFmoc-Lys(Boc) (34, 39). During all these couplings the Keiser test wasnegative and the desired backbone cyclic peptides were obtained inexcellent yields without side products in the crude. From these resultsit can be concluded that coupling with BTC under the conditionsdescribed above does not remove sensitive side chain protecting groupsnormally used in solid phase peptide synthesis with Fmoc chemistry.

We have shown that most of the Fmoc-AA chlorides do not racemize duringcouplings to building unit-peptidyl-resin in various solvents with orwithout collidine as base. The assessment of the degree of racemizationduring couplings mediated by BTC in solvent inert to this reaction isbased on the following results: (a) as shown in Table 1 whenracemization occurs, two peaks with identical mass were found by HPLC.In all the peptides shown in Table 3, including those peptides that gavelow yields (21, 23, 26, 28, 33, 36, 53, 58, 59) only the major peak gavethe desired mass. (b) coupling of Fmoc-Lys(Boc) and Fmoc-D-Lys(Boc) toPheN2 building unit-resin, further assembly of the peptides, Allyl/Allocdeprotection, cyclization and removal from the resin yielded twodiastereomeric backbone cyclic peptides (37, 38) in quantitativeconversion. The HPLC profile of each individual crude diastereomershowed a single distinct peak with the same mass and different retentiontimes from one another. Moreover, co-injection to CapillaryElectrophoresis of the two diasteromers 37 and 38 gave two separatepeaks. The lack of racemization during the coupling of Fmoc-AAschlorides to building unit-peptidyl-resin is due to the high reactivityof acid chlorides to the nucleophilic acyl substitution compared to theslower oxazolon formation. Since the BTC mediated coupling in solventsinert to this reaction proceeds via in-situ acid chloride formation, itis not surprising that generally this coupling proceeds withoutracemization.

In order to find the limitations of BTC to promote difficult couplingswe synthesized peptides 44–46 in which various Phe building units werecoupled to N-Me-Ala-peptidyl-resin. In addition, in peptide 43 Fmoc-Phewas coupled to N-Me-Phe-N-Me-Phe-resin and in peptide 48 Fmoc-N-Me-Phewas coupled to N-Me-Phe-N-Me-Phe-resin. All these repetitive couplingsproceeded in short time with quantitative conversion and leads to theconclusion that BTC is the reagent of choice to promote difficultcouplings in SPPS.

In most of the peptides presented in Table 3, the coupling was performedon di-peptidyl building unit-resin. In order to test the capability ofBTC to effect couplings to building unit attached in other positionsalong the peptide chain, coupling was performed in positions 4–6 and 11(peptides 56, 41, 22, 51 and 52, respectively).

Table 4 depicts an overview of the peptides synthesized by BTC couplingand correlates the type of the incoming building unit with the positionof coupling along the peptide chain. While couplings to positions 4–6proceeded under the normal conditions, coupling to position 11 neededsix cycles to achieve quantitative conversion.

TABLE 4 Summary of Difficult Coupling Reactions Using the BTC MethodBuilding Amino acid coupled to Building Unit Unit Ala DAla DLeu Ile LeuVal Gln Lys DLys Phg Phe PheC1 PheC2 Met Ser AlaN3 1 4a AlaC2 1 AlaC3 12 5 8 AlaC4 1 1 6 AlaC5 AlaN2 1,2 6 1,2 1 PheN2 7b 1,2 5a 1 PheC4 1PheC2 3 2 2 PheN3 LeuN4 3 2 2 2 2 LeuN3 2 LeuC3 2 LysN3 2 LysC3 2 2 2 2LysC4 6 ValC3 2 2 2 ValN3 5a 2 NMePhe 2 NMeAla 3 3 GlyC3 1 NMeGly DTrpN2Building Amino acid coupled to Building Unit Unit Arg Asp Asn His ThrNMePhe Tyr DTyr Pro Trp Cys Glu GlyC2 NMeGly GABA AlaN3 1 AlaC2 11(6),25 AlaC3 11(6) 9 AlaC4 12  AlaC5 2 AlaN2 3,5(4) 7 1 PheN2 PheC4 11  PheC22 ** 9 PheN3 1 LeuN4 1 LeuN3 4 LeuC3 LysN3 LysC3 2 2 LysC4 ValC3 # nr2*** 2 2 2 ValN3 2 MePhe 2 1 and 2 7 MeAla GlyC3 1 MeGly 2,3,4 DTrpN2 1Footnotes to Table 4: The numbers in the table indicate the buildingunit position (from C-terminal) to which the AA was coupled in >80%conversion based on HPLC and confirmed by MS *Double coupling with 5 eqAA, 1.5 eq BTC(0.33 eq per AA), 14 eq Collidine in inert solvent (0.14M) at 500 C. for 1 h **Only 20% conversion with epimerization nr = Noreaction ( ) = number of coupling cycles # = 5–10% ***= 50% apreactivation with BTSA b preactivation of the building unit-peptidylresin with BTSA, 4–7 coupling cycels and addition of AgCN

The use of BTC for the couplings of Fmoc-His(Trt) and FmocAsn(Trt) wereunsuccessful. The coupling of Fmoc-His(Trt) gave only 20% conversionwith total racemization and there was no coupling with Fmoc-Asn(Trt).

Example 62 Synthesis of Bicyclic Peptide PTR 3205

Two grams of Rink Amide (MBHA resin, NOVA, 0.46 mmol/gram) were swelledover night in NMP in a reactor equipped with a sintered glass bottom,attached to a shaker. Fmoc was removed from the resin using 25%Piperidine in NMP (16 ml) twice for 15 min. After careful wash, seventimes with NMP (10–15 ml), for 2 min each, coupling of Phe-N3 wasaccomplished using Fmoc-Phe-N3-OH (3 eq, 2.76 mmol, 1.46 gram) dissolvedin NMP (16 ml) and activated with PyBroP (2.76 mmol, 1.28 gram) and DIEA(6 eq, 5.52 mmol, 0.95 ml) for 4 min at room temperature and thentransferred to the reactor for coupling for 1 h at room temperature.Following coupling the peptide-resin was washed with NMP (10–15 ml)seven times for 2 min each. Reaction completion was monitored byqualitative Kaiser test. Fmoc removal and wash was carried out asdescribed above followed by wash with THF (10–15 ml) three times for 2min each and Fmoc-Cys(Acm)-OH (5 eq, 4.6 mmol, 1.9 gram) was coupled tothe building unit-peptidyl-resin using bis-(trichloromethyl)carbonate(1.65 eq, 1.518 mmol, 0.45 gram) and collidine (14 eq, 12.88 mmol, 1.7ml) in THF (30–35 ml, to give 0.14 M mixture) at 50° C. for 1 h. andthis coupling procedure was repeated. Assembly of Thr, Lys, (D)Trp, Phe,Cys and PheC3 was accomplished by coupling cycles (monitored byqualitative Kaiser test) using Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-(D)Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH and Fmoc-PheC3-OHrespectively, in each coupling cycle the amino acid was dissolved in NMPand was activated with PyBroP and DIEA, following coupling thepeptide-resin was washed than Fmoc removed followed by extensive washwith NMP, as described above for the first coupling. At the end of theassembly the peptidyl-resin underwent allyl/alloc deprotection under thefollowing conditions: the peptidyl resin was washed with DCM (10–15 ml)three times for 2 min each and with a mixture of DCM-AcOH-NMM (92.5%,5%, 2.5% respectively) three times for 2 min each. 3 gram of Pd(P(Ph)₃)₄were dissolved in the above mixture (80 ml) and the yellow suspensionobtained was transferred to the reactor and the mixture with thepeptidyl-resin underwent degassing (by babbling Argon through thereactor's sintered glass bottom) and then vigorously shacked for 2 h inthe dark. The peptidyl-resin washed with DCM, CHCl₃ and NMP (a total of15 washes 2 min each). Cyclization using PyBOP (3 eq, 2.76 mmol, 1.436gram) and DIEA (6 eq, 5.52 mmol, 0.95 ml) in NMP (20 ml) at roomtemperature for 1 h and then second cyclization over night (under sameconditions) took place. The peptidyl resin was washed with NMP followedby wash with DMF-water (15 ml, 4:1) three times for 2 min each. I₂solution (5 eq, 4.6 mmol, 1.16 gram) in DMF-water (23 ml, 4:1) was addedto the peptidyl-resin which was shacked at room temperature for 40 minto afford Cys-Cys cyclization. The peptidyl resin was filtered andwashed extensively with DMF/water, DMF, NMP, DCM, CHCl₃ and also with 2%ascorbic acid in DMF. After final Fmoc deprotection and wash as aboveand also wash with MeOH, followed by drying the peptidyl resin undervacuum for 20 min the peptide was cleaved from the resin using 95% TFA,2.5% TIS and 2.5% water in a total of 30 ml cocktail mixture for 30 minat 0° C. under Argon and then 1.5 h at room temperature. The solutionwas filtered through extract filter into polypropylene tube, the resinwas washed with 5–6 ml cocktail and 4–5 ml TFA, the solution wasevaporated by N₂ stream to give oily residue which on treatment withcold Et₂O solidify. Centrifugation and decantation of the Et₂O layer andtreatment with additional portion of cold Et₂O followed bycentrifugation and decantation and drying the white solid under vacuumover night gave crude PTR 3205 (0.388 gram) having the followingstructure:

Example 63 Synthesis of Tricyclic Peptide PTR 3227

PTR 3227 is a tricyclic backbone cyclic peptide having the followingstructure:

In this compound, one bridge connects a building unit (Gly-C2) to theside chain of Lys residue, a second bridge connects two building units(Phe-N2 and Phe-C2), and the third is a disulfide bridge formed betweentwo Cys residues. The synthesis procedure of this analog is nowdescribed:

1 gram Rink Amide (MBHA resin, Novabiochem, 0.46 mmol/gr) was swelledover-night in NMP in a reactor equipped with a sintered glass bottom,attached to a shaker. Fmoc was removed from the resin using 25%Piperidine in NMP (16 ml) twice for 15 min. After careful wash, seventimes with NMP (10–15 ml), for 2 min each, coupling of Lys(Dde) wasaccomplished using Fmoc-Lys(Dde)-OH (3 eq, 1.38 mmol, 0.735 gram)dissolved in NMP (8 ml) and activated with PyBroP (1.38 mmol, 0.64 gram)and DIEA (6 eq, 2.76 mmol, 0.47 ml) for 4 min at room temperature andthen transferred to the reactor for coupling for 1 h at rt. Followingcoupling the peptide-resin was washed with NMP (10–15 ml) seven timesfor 2 min each. Reaction completion was monitored by qualitative Kaisertest. Fmoc removal and wash was carried out followed by coupling ofFmoc-PheN2-OH (3 eq, 1.38 mmol, 0.71 gram) as described above. Followingcoupling Fmoc removal and wash with NMP the peptide-resin was washedwith THF (10–15 ml) three times for 2 min each and Fmoc-Cys(Acm)-OH (5eq, 2.3 mmol, 0.95 gram) was coupled to the BU-peptidyl-resin using BTC(1.65 eq, 0.759 mmol, 0.225 gram) and collidine (14 eq, 6.44 mmol, 0.85ml) in THF (16 ml, to give 0.14 M mixture) at 50° C. for 1 h. Thisdifficult coupling procedure was repeated once more. Assembly of Thr,Lys, (D)Trp, Phe, Cys and PheC2 was accomplished by coupling cycles(monitored by qualitative Kaiser test) using Fmoc-Thr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-(D)Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH andFmoc-PheC2-OH respectively, in each coupling cycle the amino acid wasdissolved in NMP and was activated with PyBroP and DIEA, followingcoupling the peptide-resin was washed than Fmoc removed followed byextensive wash with NMP, as described above for the first coupling.After the assembly of PheC2 the peptidyl-resin underwent allyl/allocdeprotection under the following conditions: the peptidyl resin waswashed with DCM (10–15 ml) three times for 2 min each and with a mixtureof DCM-AcOH-NMM (92.5%, 5%, 2.5% respectively) three times for 2 mineach. 1.5 gram of Pd(P(Ph)₃)₄ were dissolved in the above mixture (40ml) and the yellow suspension obtained was transferred to the reactorand the mixture with the peptidyl-resin underwent degassing (by babblingArgon gas through the reactor's sintered glass bottom) and thenvigorously shacked for 2 h in the dark. The peptidyl-resin was washedwith DCM, CHCl₃ and NMP (a total of 15 washes 2 min each). Cyclizationusing PyBOP (3 eq, 1.38 mmol, 0.72 gram) and DIEA (6 eq, 2.76 mmol,0.475 ml) in NMP (10 ml) at room temperature for 1 h and then secondcyclization over night (under same conditions) took place.Fmoc-deprotection was then preformed as described above followed by NMPwash, then Fmoc-GlyC2-OH was coupled to the cyclic peptide using 5 eq ofthe BU (2.3 mmol, 0.94 gram), BTC (1.65 eq, 0.759 mmol, 0.225 gram) andcollidine (14 eq, 6.44 mmol, 0.85 ml) in THF (16 ml, to give 0.14 Mmixture) at 50° C. for 1 h. and this difficult coupling procedure wasrepeated once more. Coupling of (D)-Phe to the decapeptide-resin wasdone using Boc-(D)-Phe-OH (4 eq, 1.84 mmol, 0.488 gram) and PyBrop (1.84mmol, 0.857 gram) as coupling reagent and DIEA (8 eq, 3.68 mmol, 0.63ml) as a base in DMF/DCM (1:1, 8 ml) once for 1.5 h. and second couplingcycle under same conditions for 2 h at room temperature. Next, followedwash selective removal of Dde from Nε side chain of the Lys (at position1 from C-terminal) was done using 2% hydrazine in DMF (25 ml×3 min×3) atrt. after wash with DMF and NMP the peptidyl resin underwent allyldeprotedtion as described above using 0.75 gram of Pd(P(Ph)₃)₄. Thepeptidyl resin was washed with CHCl₃, DCM and NMP followed bycyclization with PyBOP as described above (Kaiser test was negative).Followed by wash with DMF-water (12.5 ml, 4:1) three times for 2 mineach. I₂ solution (5 eq, 2.3 mmol, 0.583 gram) in DMF-water (12.5 ml,4:1) was added to the peptidyl-resin which was shacked at roomtemperature for 40 min to afford Cys-Cys cyclization. The peptidyl resinwas filtered and washed extensively with DMF/water, DMF, NMP, DCM, CHCl₃and also with 2% ascorbic acid in DMF followed by DMF. After wash withDCM and MeOH, followed by drying the peptidyl resin under vacuum for 20min the peptide was cleaved from the resin using 95% TFA, 2.5% TIS and2.5% water in a total of 15 ml cocktail mixture for 30 min at 0° C.under Argon and then 1.5 h at room temperature. The solution wasfiltered through extract filter into polypropylene tube, the resin waswashed with 5–6 ml cocktail and 4–5 ml TFA, the solution was evaporatedby N₂ stream to give oily residue which on treatment with cold Et₂Osolidify. Centrifugation and decantation of the Et₂O layer and treatmentwith additional portion of cold Et₂O followed by centrifugation anddecantation and drying the with solid under vacuum over night gave crudePTR-3227 (72 mg, 10%). The HPLC chromatogram of the crude peptide isrepresented in FIG. 5.

Example 64 Coupling of Galactose Derivative to a Backbone Cyclic Peptideto Yield PTR 3229

Backbone cyclic peptide underwent Fmoc deprotection followed by washeswith NMP and THF. A coupling cycle with 1,2:3,4-Di-O-isopropylideneD-galactopyranose (5 eq, 1.15 mmol, 0.3 gram) using 0.33 eq of BTC(0.379 mmol, 0.112 gram) as the coupling reagent and 14 eq of collidine(3.22 mmol, 0.425 ml) as a base in THF (15 ml) at rt was preformed for 1h. After wash with DCM and MeOH the peptide was cleaved from the resinusing TFA (95%), TIS (2.5%) and water (2.5%) in a total volume of 15 mlcocktail mixture for 1.5 h. at rt. After further work-up crude PTR-3229(0.176 gram), having the sequence:Galactose-Dab-Phe-Trp-(D)Trp-Lys-Thr-Phe-Gly(C3)-NH₂, was obtained Thispeptide analog comprises a bridge connecting the GlyC3 building unit andthe Dab residue. The mass spectra analysis of the crude peptide arerepresented in FIG. 6.

Example 65 Formation of a Urea Bond Using BTC

-   A. Formation of a urea bond in the bridge by coupling Ile    (isocyanate derivative formed by BTC), to Lys-peptidyl resin to form    the peptide analog PTR 3237 having the following structure:

-    The mass spectra analysis of the crude peptide are represented in    FIG. 7.-   B. Formation of urea bond in the sequence by using diamine as    follows: To 5 eq of mono Fmoc-diamine hydrochloride in CH₂Cl₂    (0.15M), 1 eq DIEA (for neutralization of the hydrochloride) was    added. After 1 min 1.65 eq of BTC were added and the suspension was    stirred for 2 minutes, followed by addition of 14 eq of DIEA. After    1 minute, the resulted solution was added to the peptidyl resin and    the mixture was agitated for 1 hour at room temperature followed by    CH₂Cl₂ and NMP washes. The resulted backbone cyclic peptide analog    denoted PTR 3241 has the following structure:

-    The HPLC chromatogram and the mass spectra analysis of the crude    peptide are represented in FIG. 8.

Example 66 Multiple Parallel Synthesis Using BTC

About 500 separate peptides were synthesized in MPS format using the BTCas coupling reagent. An example of synthesis results of 24 peptides issummarized in table 5.

TABLE 5 MS qualitative Calc. results Found SEQ ID NO:5 1 1 a GlyN3 ArgVal Gln AlaC2 Phe Thr 888.41 (++) 889.2 SEQ ID NO:6 2 1 b GlyN3 Arg ProGln PheC2 Phe Thr 962.42 (++) 963.1 SEQ ID NO:7 3 1 c GlyN3 Ala Val GlnGlyC2 Phe Thr 789.33 (++) 790.1 SEQ ID NO:8 4 1 d GlyN3 Tyr Ala GlnLysC3 Phe Thr 938.39 (+−) 938.9, 462.9(cyclic tripep) SEQ ID NO:9 5 1 eGlyN3 Thr Ser Gln LeuC3 Phe Thr 877.36 (++) 878.1 SEQ ID NO:10 6 1 fGlyN3 Gly Gly Gln ValC3 Phe Thr 789.31 (+−) 790, 434(cyclic tripep) SEQID NO:11 7 1 g GlyN3 Ala Ala Gly AlaC2 Phe Thr 704.28 (++) 705 SEQ IDNO:12 8 1 h GlyN3 Tyr Gly Gly PheC2 Phe Thr 858.31 (++) 859 SEQ ID NO:139 2 a GlyN3 Ile Arg Gly GlyC2 Phe Thr 817.36 (++) 818 SEQ ID NO:14 10 2b GlyN3 Leu Met Gly LysC3 Phe Thr 877.37 (++) 878.1 SEQ ID NO:15 11 2 cGlyN3 Lys Asp Gly LeuC3 Phe Thr 861.36 (++) 862 SEQ ID NO:16 12 2 dGlyN3 Met Ala Gly ValC3 Phe Thr 806.31 (++) 806.9 SEQ ID NO:17 13 2 eGlyN3 Phe Tyr Ile AlaC2 Phe Thr 928.39 (++) 929 SEQ ID NO:18 14 2 fGlyN3 Pro Ile Ile PheC2 Phe Thr 904.42 (+−) 905, 553(tripep + piper) SEQID NO:19 15 2 g GlyN3 Ser Gly Ile GlyC2 Phe Thr 748.29 (++) 748.9 SEQ IDNO:20 16 2 h GlyN3 Thr Asp Ile LysC3 Phe Thr 905.39 (−) 463(tripep +piper) SEQ ID NO:21 17 3 a GlyN3 Trp Asp Ile LeuC3 Phe Thr 975.41 (−)975.9, 448(tripep + piper), 893.9(?) SEQ ID NO:22 18 3 b GlyN3 Tyr GlyIle ValC3 Phe Thr 880.37 (−) 881, 434(tripep + piper), 865.9(M-NH3) SEQID NO:23 19 3 c GlyN3 Val Tyr Leu AlaC2 Phe Thr 880.39 (++) 881 SEQ IDNO:24 20 3 d GlyN3 Phe Asp Leu PheC2 Phe Thr 956.39 (++) 956.9 SEQ IDNO:25 21 3 e GlyN3 Ile Asp Leu GlyC2 Phe Thr 832.35 (++) 832.9 SEQ IDNO:26 22 3 f GlyN3 Phe Gln Leu LysC3 Phe Thr 964.44 (++) 964.9 SEQ IDNO:27 23 3 g GlyN3 Glu Ser Leu LeuC3 Phe Thr 890.37 (++) 891 SEQ IDNO:28 24 3 h GlyN3 Gln Pro Leu ValC3 Phe Thr 885.4 (++) 886 Notes toTable 5: Difficult coupling procedure: pre-activation of the AA outsideof an automatic peptide synthesizer (ACT 396 with Labtech 4 fromAdvanced ChemTech). Five eq AA in 150 ul dioxane + 1.65 eq BTC in 150 uldichloropropane, mixing, after 1 min 14 eq collidine in dichloropropanewere added. The pre-activated amino acids were transferred to the platemanually. Reaction for 3 times 1 h at 60° C. HPLC chromatogram and massspectra analysis examples of crude MPS peptide (number 20 in table 5),are represented in FIG. 9.

Example 67 Synthesis of a Cyclosporine Analog Using BTC

The BTC reagent was used for the difficult couplings in the synthesis ofa Cyclosporine analog having the sequence:

Ala-NMeLeu-NMeLeu-NMeVal-NMeLeu-Abu-Sar-NMeLeu-Val-NMeLeu-Ala-NH₂

This peptidemimetic is a linear derivative of the Cyclosporin analogdescribed in Raman et al. J. Org. Chem. 63:5734, 1998. The HPLCchromatogram and mass spectra analysis of the crude peptide arerepresented in FIG. 10.

1. In the coupling of a carbohydrate moiety to a peptide, theimprovement which comprises forming a bond between the peptide and thecarbohydrate moiety by in-situ reaction usingbis-(trichloromethyl)carbonate, diphosgene or phosgene.
 2. The method ofclaim 1, wherein bis-(trichloromethyl)carbonate is used to form thebond.
 3. The method of claim 1, wherein diphosgene is used to form thebond.
 4. The method of claim 1, wherein phosgene is used to form thebond.
 5. The method of claim 1, wherein the peptide reacts on the aminoterminus to form the bond.